A Guide to Nucleic Acid Labeling and Detection Systems

A GUIDE TO NUCLEIC ACID LABELING AND DETECTION SYSTEMS

VECTOR LABORATORIES S T A T E O F T H E A R T L A B E L I N G & D E T E C T I O N S Y S T E M S 1

Table of Contents

Introduction ...... 2-3

Labeling of Nucleic Acids ...... 4-9

Choosing a Labeling System Based on Nucleic Acid Type and Size...... 4 Choosing a Labeling System Based on Application and Label...... 5 PHOTOPROBE ® Reagents and the FastTag ® System...... 6-7 5’ EndTag ™ and 3’ EndTag ™ Labeling Systems...... 8-9

Detection of the Nucleic Acid Label ...... 10-13 Antibody Conjugates...... 10 Complementary Detection Reagents...... 11-13 Fluorescent Systems...... 11 VECTASHIELD ® Mounting Media...... 12 Chromogenic Substrates for Enzyme-Based Detection...... 13 DuoLuX ™ Chemiluminescent/ Fluorescent Substrate...... 13

Applications ...... 14-18 Southern and Northern Blot Hybridization...... 14 In Situ Hybridization...... 15 Optimizing Probe Length for In Situ Hybridization...... 15 Comparative Genomic Hybridization...... 15 Cellular Localization...... 16 EndTag ™ - Labeled Primers Used in PCR Amplification...... 16 Covalent Attachment of 5’ EndTag ™ -Labeled Nucleic Acids to Gold...... 16 Affinity Binding...... 17 Resolve Same-Sized DNA in a Sequence-Specific Manner...... 18 Amplification of Fluorescent Signal in Nucleic Acid Microarrays...... 18

Protocols ...... 19-26

Assessing Labeling Efficiency - Estimation of Labeling by Comparative Dot Blot...... 19 Assessing Labeling Efficiency - Quantitation of Biotin Using the Quant*Tag ™ Biotin Kit...... 19 Southern Blot Hybridization and Detection...... 20-21 FISH: Fluorescence Detection of Biotin-Labeled ISH Probes...... 22 FISH: Fluorescence Detection of Fluorescein-Labeled ISH Probes...... 23 ISH: Chromogenic Detection of Biotin-Labeled ISH Probes...... 24 ISH: Chromogenic Detection of Fluorescein-Labeled ISH Probes...... 25 Methods used to elute single strands of DNA from VECTREX ® - bound biotinylated DNA...... 26 Nucleic Acid Purification ...... 27

Fast Facts ...... 28

References ...... 29-31

Ordering Information ...... 32

Inside front cover : A plasmid biotinylated with the FastTag ® Biotin Labeling System was complexed with a lactosylated polyethylenimine and instilled intranasally to mice. On this tissue slide, the plasmid labeled with rhodamine-conjugated streptavidin (red) is detected in ep ithelial cells of the bronchus and in the pulmonary cells surrounding it. Image kindly supplied by G.Thévenot and Dr. I. Fajac, Faculté de Médecine Paris 5, Paris, France. 2

Introduction Detection of Nucleic Acid Labels

Recognized as a leader in the development of Each choice of label is complemented by several de tection systems, Vector Laboratories has been different fluorescent or enzyme-based detection providing the research community with quality reagents providing the researcher with flexibility reagents yielding high sensitivity and low back - in optimizing experimental systems. Among the ground since 1976. Continuing in this tradition, ex tensive range of antibody conjugates and our molecular biology tools offer sensitivity, avidin/streptavidin conjugates, amplification relia bility, simplicity, and economy. These systems reagents such as Biotinylated Anti-Streptavidin and and reagents facilitate the labeling, detection, Biotinylated Anti-Avidin are also available. These and manipulation of nucleic acids. Microarrays, reagents reliably increase sensitivities of target blotting assays, cell trafficking, in situ hybridization, sig nals in microarray applications and fluorescence comparative genomic hybridization, subtractive in situ hybridization. In addition, the superior anti- hy bridization, and affinity purification are just a fading property of the VECTASHIELD ® line of few applications in which our molecular biology mounting media ensures the preservation of signal tools can be successfully employed. intensity in applications such as fluorescence in situ hybridization. (Please see pages 10-13 for a listing Labeling of Nucleic Acids of selected products.)

The PHOTOPROBE ® reagents, FastTag ® Systems, For nucleic acid blotting applications, the Vector ® and the 3’ and 5’ EndTag ™ Labeling Systems are UltraSNAP ™ Detection System (page 14) containing efficient and reliable labeling methods that specially formulated reagents detects biotinylated offer choices for optimum labeling for a given probes with high sensitivity and low background. ap plica tion, as well as choices for incorporating This system utilizes the DuoLuX ™ Chemiluminescent/ fluorochromes, haptens, or affinity tags. Fluorescent Substrate with in tense and prolonged light emission characteristics, ideal for Southern and Nucleic acid probes for applications such as in situ northern hybridization, dot blot, plaque or colony hybridization, northern and Southern blot screening. hy bridization, comparative genomic hybridization, intracellular localization experiments, and DNA microarrays can be easily and optimally labeled and subsequently visualized with our comprehen - sive range of fluorescent, chemiluminescent, or chromogenic detection systems.

FastTag ® Fluorescein labeled pUC1.77 (yellow-green) detected Cellular uptake of labeled plasmid: FastTag ® Fluorescein labeled with Biotinylated Anti-Fluorescein and Fluorescein Avidin DCS. plasmid DNA (green) incubated with COS-7 cells. Nuclei were Nuclei were counterstained and mounted with VECTASHIELD ® counterstained and mounted with VECTASHIELD ® with DAPI with PI (red-orange). (blue).

www.vectorlabs.com 3

Immobilization of Labeled Probes Quantitation of Biotin using the Quant *Tag ™ System

Vector Laboratories offers both irreversible The Quant*Tag ™ Biotin Kit (page 19) is designed to (VEC TREX ® Avidin D) as well as reversible determine the amount of free biotin in solution or (VECTREX ® Avidin DLA or VECTREX ® AAL) affinity the number of biotins attached to proteins, nucleic binding matrices. Applications like genomic/ cDNA acids or other macromolecules. Samples do not subtraction or library screening by hybrid capture need to be predigested. The kit reagents chemically require labeled nucleic acids to be immobilized react with free or bound biotin, producing a colored onto a solid support. Using our affinity binding product that can be quantified using a spectropho - matrices in such applications can yield information tometer. The absorbance is measured in the visible about differential gene expression or allow the spectrum, allowing the use of plastic cuvettes or iso lation and subsequent amplification of a gene of microtitre plates. interest. (Please see page 17 for further information).

Electrophoretic Resolution of Similarly Sized DNA in a Sequence Specific Manner

The Resolve -It ™ Kit (page 18) contains two sequence-specific DNA ligands which bind to DNA and retard its electrophoretic migration in a sequence specific manner. The separation of the DNA species allows for the gel purification of DNA samples which would otherwise be very difficult to isolate. The ability to resolve DNA in a gel is critical for applications such as differential display in which multiple DNA species of similar size need to be separated before subsequent excision and analysis.

Detection by electron microscopy of a plasmid biotinylated with the FastTag ® Biotin Labeling System and then gold-labeled. The plasmid, complexed with lactosylated PEI, is taken up by an airway epithelial cell. Image kindly supplied by Drs. S. Grosse and I. Fajac, Faculté de Médecine Paris 5, Paris.

Detection in an airway epithelial cell of a plasmid biotinylated with the FastTag ® Biotin Labeling System and then labeled with rhodamine-conjugated streptavidin (red). The nuclear membrane is labeled with an anti-lamin A/C antibody and ap pears green. Image kindly supplied by Drs. S. Grosse and I. Fajac, Faculté de Médecine Paris 5, Paris. 4

Labeling of Nucleic Acids

Two important considerations in determining what Thus, the PHOTOPROBE ® or FastTag ® chemical labeling system will work best in a given applica - labeling method is especially convenient for la bel - tion are: (1) size and type of the nucleic acid to be ing samples that will be used to observe cellular labeled; (2) choice of label required for the appli - lo calization of nucleic acid (e.g. plasmid DNA in cation. gene delivery or siRNA) or quantitative comparison.

Size and type. Longer strands of DNA (>100 bp) or Shorter strands of nucleic acids such as oligonu - circular DNA (i.e. plasmid DNA), RNA or PNA (pep - cleotides, PCR primers, or capture probes used to tide nucleic acid) used in blots, in situ hybridization, identify nucleic acid binding proteins are specifically subtractive hybridization, or other procedures are and efficiently labeled at either the 5’ or the 3’ end efficiently and reliably labeled with the PHOTO - using the 5’ E’ ndTag ™ or the 3’ E’ ndTag ™ Nucleic Acid PROBE ® labeling reagents or the FastTag ® Nucleic Labeling Systems, respectively. The 5’ E’ ndTag ™ Acid La beling System. Either labeling method re - or the 3’ E’ ndTag ™ Kits can be used to attach a sin gle sults in the covalent coupling of the label to the fluorochrome or affinity tag at the appropriate sample. These systems ensure multiple site labeling end of nucleic acids. 5’ E’ ndTag ™ Labeling kit uses over the entire length of the nucleic acid resulting both DNA and RNA as a substrate whereas the 3’ in greater accessibility of the affinity tag or greater E’ ndTag ™ Kit will selectively label only DNA. sensitivity in detection. Application. The second important factor in deter - The integrity of the nucleic acid is preserved in mining the most appropriate labeling system is the this non-destructive reaction making it useful for choice of label, hapten, or affinity tag required for applications where it is necessary to use the intact, the given application. The PHOTOPROBE ® reagents original sample. With the PHOTOPROBE ® or FastTag ® incorporate a specific tag (e.g. biotin) into nucleic labeling systems, the entire length of the original acid in one simple step. The versatility of the FastTag ® nucleic acid sample, rather than copies, is directly, Labeling kit, 5’ EndTag ™ or 3’ EndTag ™ Labeling labeled. In contrast, enzymatic labeling methods Kits allows a variety of tags to be incorporated. such as random priming or nick translation are Some com monly used labels that are available dif ficult to control and don’t label the original from Vector Laboratories are briefly described as nu cleic acid sample. Instead, these methods result in fol lows. Detection reagents for these labels are a labeled copy produced from the original template. listed on page 10.

Choose a Labeling System based on Nucleic Acid Type and Size

www.vectorlabs.com 5

Biotin - Because of the extraordinary affinity of Fucose - This unique label is ideal for reversible binding avidin and streptavidin to biotin and the many of labeled nucleic acid to the matrix, VECTRE X® AAL. biotin-avidin/streptavidin systems available, this Fu cose labeled nucleic acids can be bound and eluted label is ideal for a variety of applications including under mild conditions. (For more information, please see in situ hybridization, blotting, and affinity binding. page 17). Alkaline phosphatase Aleuria aurantia lectin can be used in dot blot applications to assess labeling DNP (Dinitrophenyl) - Because DNP is not found efficiency with the fucose label. endogenously in tissue, it is an excellent alter native Texas Red ® - This fluorescent label is a high to biotin. H igh affinity, purified antibodies are quantum yield rhodamine derivative that can be available for detection or amplification of the directly visualized. The label can also be signal. detected, and the signal ampli fied, using our affinity-purified antibody conjugates to rhodamine. Fluorescein - This fluorescent label can be directly (Excitation maximum at 595 nm; emission maximum detected or used as a hapten and detected with at 615 nm). our high affinity antibody reagents. (Excitation maximum at 495 nm; emission maximum at 515 nm).

Choose a Labeling System based on Application and Label 6

PHOTOPROBE ® Reagents and A distinct advantage of the simple setup in each of these labeling options is the ease and economy of ® the FastTag System scaling up.

PHOTOPROBE ® Reagents and the FastTag ® Nucleic PHOTOPROBE ® Biotin incorporates biotin in one Acid Labeling Systems are the methods of choice re action step. PHOTOPROBE ® (Long Arm) Biotin for incorporating a label at multiple sites along has an extra long linker arm and should be used if the entire length of the nucleic acid. The labeling increased distance between the sample and the reaction does not destroy the original nucleic acid tag is needed. In a similar manner, PHOTOPROBE ® nor creates its copy. The integrity of the original Amine incorporates primary amines into nucleic sample is preserved. Single- 15, 28 or double- acids. These amino groups can subsequently be used stranded 11,20 DNA, circular DNA, 45, 58 RNA, 22 siRNA, 49 to attach haptens, affinity tags, or fluorochromes, or or PNA (peptide nucleic acid) can be labeled with to immobilize nucleic acids to a solid matrix. the same reagents. The FastTag ® Nucleic Acid Labeling System utilizes Labeling with either system is based on aryl azide a disulfide-containing universal linker that is incor - chemistry in which either reagent, when exposed porated into nucleic acid simply by using heat or to heat or light, becomes activated and incorpo - light. Reducing the disulfide bond yields a free rates into the nucleic acid without base specificity. thiol group. A variety of thiol-reactive haptens, The labeling reaction can be carried out using a fluorochromes, affinity ligands or other markers mercury vapor bulb (sun lamp), a UV lamp which can then be covalently bound to the nu cleic acid produces light in the 350 nm to 370 nm range, a via the FastTag ® reagent thiol group. See page 7 halogen lamp, a heating block, or a thermal cycler for a list of thiol-reactive reagents available from providing the investigator with great flexibility in Vector Laboratories. experimental design.

PHOTOPROBE ® FastTag ®Labeling Reaction Labeling Reaction S S

LIGHT or HEAT 30 min LIGHT 1 or S HEAT 10-30 min S

PHOTOPROBE ® labeling (above) occurs in one short heat- or photo- labeling step. REDUCING 2 REAGENT HS 10 min SH FastTag ® labeling (right) occurs in three easy steps:

Step 1 . The FastTag ® Universal Linker is incorporated into the nucleic acid upon exposure to heat or light. O N O Step 2. The disulfide bond is reduced, yielding a free thiol group. O N O Step 3 . A covalent bond is formed between the FastTag ® reagent 3 THIOL- thiol and a thiol-reactive hapten, fluorochrome, affin ity ligand, REACTIVE S LABEL or other marker. 10 min at 65 °C or 30 min at rt

www.vectorlabs.com 7

Each PHOTOPROBE ® Biotin Kit contains the Labeling Systems following components to label up to 250 µg of nucleic acid (or to carry out up to 50 labeling FastTag ® Labeling Kit MB-8000 • 1 Kit reactions): PHOTOPROBE ® Biotin SP-1000 • 0.5mg PHOTOPROBE ® (Long Arm) Biotin • PHOTOPROBE ® Biotin Reagent SP-1020 • 0.5mg • Tris Buffer PHOTOPROBE ® Biotin • sec-Butanol Labeling and Detection System • Biotinylated DNA Standard SPK-1906 • 1 Kit • Precipitant PHOTOPROBE ® Amine SP-1070 • 0.5mg

The PHOTOPROBE ® Amine Kit includes the following components to label up to 360 µg of Thiol-reactive Labeling Reagents nucleic acid (or to carry out up to 72 labeling reactions): Biotin Maleimide SP-1501 • 12mg DNP Maleimide SP-1503 • 1mg • PHOTOPROBE ® Amine Fluorescein Maleimide SP-1502 • 12mg • Borate Buffer Fucose Maleimide SP-1504 • 500µg • sec-Butanol Texas Red ® Maleimide SP-1505 • 3.6mg • Precipitant These thiol-specific labels can be used with the FastTag ® The FastTag ® Labeling Kit † contains the following Labeling Kit, 5’ EndTag ™ or 3’ EndTag ™ Labeling Kits. reagents to label up to 250 µg of nucleic acid (or to carry out up to 50 labeling reactions):

• FastTag ® Reagent • Tris Buffer • Citrate Buffer • Reducing Reagent • sec-Butanol • Precipitant

†Use this kit with any thiol-reactive label.

Aryl Azide Chemistry 8

5’ EndTag ™ and 3’ EndTag ™ The 5’ EndTag ™ system labels 5’ ends of DNA, RNA, 30, 60 or unmodified oligonucleotides. 47, 57 Labeling Systems 3’ end labeling is preferred over 5’ end labeling if End labeling is a favored method for applications the terminal phosphate at the 5’ end must be pre - where an internal label might interfere with served. 3’ EndTag ™ labeled nucleic acids can be hy bridization or sequence-specific protein bind ing. used for applications such as DNA hybridization, Short oligonucleotides are labeled more efficiently PCR, in situ hybridization, or EMSA. The 3’ EndTag ™ with these systems than with other methods. In system enables simple and uniform labeling of 3’ addition, end labeling of oligonucleotides is an ends of DNA. economical alternative to having labels inserted during synthesis. 5’ EndTag ™ Kit MB-9001 • 1 Kit 3’ EndTag ™ Kit MB-9002 • 1 Kit Both the 5’ EndTag ™ and the 3’ EndTag ™ Nucleic Acid Labeling Systems enable the covalent attach - A thiol-reactive label is not included in the kit, but one ment of a variety of fluorescent dyes, haptens or can be selected from the list below. affinity tags to the respective ends of the nucleic acids using thiol-specific chemistry. Thiol-reactive Labeling Reagents

5’ EndTag ™ is ideal for labeling PCR primers because Biotin Maleimide SP-1501 • 12mg a label is attached only at the 5’ end, leaving the DNP Maleimide SP-1503 • 1mg 3’ end available for polymerization. The end Fluorescein Maleimide SP-1502 • 12mg posi tion of the label generally does not interfere Fucose Maleimide SP-1504 • 500µg ® with hybridization or nucleic acid binding and is, Texas Red Maleimide SP-1505 • 3.6mg therefore, appropriate for binding of capture probes to affinity matrices 30, 52 and for electrophoretic mo bility shift assays (EMSA). 19, 37

Transmission electron micrographs of molecules of influenza A viral ribonucleoprotein particles (vRNPs) labeled at the 5’ end of the vRNA with biotin using the 5’ EndTag ™ Kit, and further - energy + energy labeled with streptavidin gold. vRNPs from influenza A were - cytosol + cytosol pu rified according to Kemler et al., (1994 Virology, 2 02:1028- 1033) on a glycerol gradient with modifications as de scribed in Wu et al., (2007, Virol J. Jun 4;4:49). Courtesy of Drs. Winco WH Nuclear import assay in digitonin-permeabilized HeLa cells of Wu and Nelly Panté, University of British Columbia, Vancouver biotinylated vRNPs. vRNPs were labeled first at the 5’ end of BC, Canada. the vRNA with biotin using the 5’ EndTag ™ Kit, and then with Vector ® Fluorescein Streptavidin. This allowed for direct fluores - cence visualization of the vRNPs on a confocal fluorescence mi croscope. Nuclear import assays were carried out as described in Wu et al., (2007, Virol J. Jun 4;4:49). The negative control con - sists of vRNPs added to the cells in the absence of energy and ex ogenous cytosol. In the presence of energy and cytosol, the fluorescein-labeled vRNPs successfully enter the nucleus, with a high degree of nucleolar staining. Courtesy of Drs. Winco WH Wu and Nelly Panté, University of British Columbia, Vancouver BC, Canada.

www.vectorlabs.com 9

5’ End Labeling Kit 3’ End Labeling Kit

Labeling with the 5’ EndTag ™ Kit is achieved by Labeling using the 3’ EndTag ™ Labeling Kit is a simple two step process. The first step is to achieved in two steps. The first step is to incorporate in cor porate a thiol group onto the 5’ end of the a thiol group onto the 3’ end of the DNA. This is oligonucleotide. This is accomplished by T4 accomplished by using a terminal transferase enzyme polynucleotide kinase which transfers a thiol- (TdT) to attach SH-GTP, a modified guanosine phos phate from ATP γS to the 5’-OH group of the triphosphate containing a thiol, to the double or oligonucleotide. In the second step the thiol- single stranded DNA end. Blunt, overhanging, con taining oligonucleotide is incubated with a or recessed 3’-OH ends may also be used. In the thiol-reactive label (e.g. biotin-maleimide). Labeling second step the thiol-containing oligonucleotide is requires about 1 hour with very few minutes of incubated with a thiol-reactive label (e.g. biotin hands-on time. maleimide). Labeling time is about 1 hour with very little hands-on time. Labeling efficiency is The 5’ EndTag ™ Kit is designed to perform 10 labeling equivalent to that achieved with traditional methods reactions of up to 0.6 nmols of 5’ ends (e.g. about but the 3’ EndTag ™ labeling system offers greater 5 µg of a 25 base oligo) per reaction and includes: versatility in the choice of label at a fraction of the cost. • T4 polynucleotide kinase • 10x reaction buffer The 3’ EndTag ™ Labeling Kit is designed to perform • ATP γS 20 labeling reactions of up to 0.5 nmols of 3’ ends • Precipitant (e.g. about 3.3 µg of a 20 base oligonucleotide) per • Alkaline Phosphatase reaction and includes:

(A thiol-reactive label is not included.) • Terminal transferase (TdT) • SH-GTP Figure A schematically shows the 3’ EndTag ™ labeling • 10x TdT buffer reaction. Figure B shows the labeling reaction for • Precipitant the 5’ EndTag ™ Labeling System. (A thiol-reactive label is not included.)

≈ SH ≈ ≈

P PP P P P O N O O N O SH-GTP SH S P TdT P Maleimide-label P

P P OH 30 minutes, 30 minutes, 37 ºC 65 ºC

O- O N O O- 5’ HO HS-P-O S-P-O O O P P P

O N O P P P Kinase, ATP S Maleimide label P γ P P 30 minutes, 30 minutes, 37 °C 65 °C 10

Detection of the Nucleic Acid Label

Our range of products offers various options for DNP (Dinitrophenyl) - Because DNP is not found detection of a given label. To select the desired endogenously in tissue, it is an excellent alter native vi sualization method, choose the reagent according label to biotin. H igh affinity, purified antibodies to the label being detected. Then choose the are available for detection or amplification of the complementary visualization reagents, either signal. fluo rescence or enzyme-based, from the lists on the following pages. Alkaline Phosphatase Anti-DNP made in rabbit MB-3100 • 150µg All antibodies to the labels are affinity-purified Biotinylated Anti-DNP and are ideal for detecting labeled probes in such made in rabbit BA-0603 • 0.5mg applications as in situ hybridization (please see pages 22-25 for protocols) or northern and South - Fluorescein - This fluorescent label can be directly ern blotting (please see pages 20-21 for protocol). detected or used as a hapten and detected with any of our high affinity antibody reagents. (Excita - Biotin - Because of the many biotin-avidin/strept- tion maximum at 495 nm; emission maximum at avidin systems available, this label is ideal for a 515 nm). variety of applications including in situ hybridization, blotting, and affinity binding. Anti-Fluorescein SP-0601 • 1mg made in goat Alkaline Phosphatase Anti-Biotin Alkaline Phosphatase Anti-Fluorescein made in goat SP-3020 • 1ml made in goat MB-2100 • 150µg Alkaline Phosphatase Streptavidin Peroxidase Anti-Fluorescein SA-5100 • 1ml made in goat SP-1910 • 0.5mg Peroxidase Anti-Biotin Biotinylated Anti-Fluorescein made in goat SP-3010 • 1mg made in goat BA-0601 • 0.5mg Peroxidase Streptavidin SA-5004 • 1mg Fucose - This unique label is ideal for reversible binding VECTASTAIN ® ABC Kit of labeled nucleic acid to the matrix, VECTRE X® AAL. (Standard) PK-6100 • 1 kit Fu cose labeled nucleic acids can be bound and eluted Fluorescein Anti-Biotin under mild conditions. (For more information, please see made in goat SP-3040 • 0.5mg page 17). Alkaline phosphatase Aleuria aurantia lectin Fluorescein Avidin DCS A-2011 • 1mg can be used in dot blot applica tions to assess labeling Fluorescein Streptavidin SA-5001 • 1mg efficiency with the fu cose label. Texas Red ® Avidin DCS A-2016 • 1mg Alkaline Phosphatase Aleuria aurantia Texas Red ® Streptavidin SA-5006 • 1mg lectin (AAL) MB-4100 • 150µg AMCA Avidin D A-2008 • 5mg Biotinylated Aleuria aurantia lectin (AAL) AMCA Streptavidin SA-5008 • 1mg made in goat B-1395 • 1mg Phycoerythrin Avidin D A-2007 • 1mg Phycoerythrin Streptavidin Texas Red ® - This fluorescent label is a rhodamine SA-5007 • 1mg derivative that can be directly visualized. The label Anti-Biotin-M (monoclonal) can also be detected, and the signal amplified made in mouse MB-9100 • 1ml using our affinity-purified antibody conjugates to Anti-Biotin (polyclonal) rhodamine. (Excitation maximum at 595 nm; made in goat SP-3000 • 1mg emis sion maximum at 615 nm). Biotinylated Anti-Avidin made in goat BA-0300 • 0.5mg Alkaline Phosphatase Anti-Rhodamine* Biotinylated Anti-Streptavidin made in goat MB-1920 • 150µg made in goat BA-0500 • 0.5mg Biotinylated Anti-Rhodamine* UltraSNAP ™ Detection Kit made in goat BA-0605 • 0.5mg for blots MB-6500 • 1 Kit *Binds most rhodamines including Texas Red ®

www.vectorlabs.com 11

Complementary Detection Reagents

Fluorescent Systems

The following fluorescent detection reagents are ideal for applications such as fluorescence in situ hybridization. Biotinylated Anti-Avidin D 5, 18, 29 and Biotinylated Anti-Streptavidin 56 are ef fectively used for amplifying fluorescent sig nals in biotin- avidin/streptavidin systems (please see page 22 for protocol).

After choosing a fluo rescent detection method, choose one of the VECTASHIELD ® mounting media on page 12 for mounting and coverslipping. VEC TASHIELD ® mounting media are unsurpassed in preserving and preventing the fading of fluorescently labeled sections, cells or spreads.

Fluorescein Avidin DCS A-2011 • 1mg Fluorescein Streptavidin SA-5001 • 1mg Texas Red ® Avidin DCS A-2016 • 1mg Texas Red ® Streptavidin SA-5006 • 1mg AMCA Avidin D A-2008 • 5mg AMCA Streptavidin SA-5008 • 1mg Phycoerythrin Avidin D A-2007 • 1mg Phycoerythrin Streptavidin SA-5007 • 1mg Biotinylated Anti-Avidin* made in goat BA-0300 • 0.5mg Biotinylated Anti-Streptavidin* made in goat BA-0500 • 0.5mg Fluorescein Anti-Goat IgG made in rabbit FI-5000 • 1.5mg Fluorescein Anti-Mouse IgG made in horse FI-2000 • 1.5mg Texas Red ® Anti-Goat IgG Coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (TPF) imaging of fluorescein-labeled HCV RNA made in rabbit TI-5000 • 1.5mg in side Huh-7 cells. HCV RNA in-vitro transcripts were labeled Texas Red ® Anti-Mouse IgG with a fluorescein-maleimide label (SP-1502) using the 5’ EndTag ™ Nucleic Acid Labeling System. Huh-7 cells were transfected with made in horse TI-2000 • 1.5mg 3 µg of 5’-fluorescein-labeled HCV RNA and imaged at 4 h post- AMCA Anti-Goat IgG transfection. Top: CARS and TPF of Mock transfected Huh-7 cells. Bottom: CARS and TPF of Huh-7 cells transfected with fluore- made in rabbit CI-5000 • 1.5mg scein-labeled HCV RNA. Figure courtesy of Jennifer Haley, Sylvie AMCA Anti-Mouse IgG Belanger, Adrian Pegoraro, Albert Stolow, and John P. Pezacki, The Steacie Institute for Molecular Sciences, The National made in horse CI-2000 • 1.5mg Re search Council of Canada. Unpublished data.

* Amplifying Reagent

FLUOROPHOR EXCITATION EMISSION AMCA 350 nm 450 nm Fluorescein 495 nm 515 nm Texas Red ® 595 nm 615 nm Phycoerythrin 450-570 nm 574 nm 12

VECTASHIELD ® Mounting Media Both the VECTASHIELD ® Hard !Set ™ and the original VECTASHIELD ® Mounting Media are available with ® VECTASHIELD Mounting Media are unsurpassed or without the counterstain DAPI (4,6’diamidino- in preventing the rapid loss of fluorescence during 2-phenylindole; blue). The original VECTASHIELD ® microscopic examination. The different formulations Mounting Medium is also available with the coun - ® of VECTASHIELD Mounting Media all offer the same terstain propidium iodide (PI; red). These counter - outstanding anti-fade and anti-photobleaching stains are provided at an optimal concentration of properties. They are all compatible with fluorescein, 1.5 µg/ml. Lower concentrations can be achieved ® Texas Red , AMCA and a wide variety of other fluo - by dilutions with the appropriate VECTASHIELD ® rochromes. Different formulations are available Mounting Medium. to best suit the investigators’ priorities and experi - mental design. DAPI produces a blue fluorescence when bound to DNA with exci tation at about 360 nm and emission ® The original VECTASHIELD Mounting Medium is a at 460 nm. PI has a broad excitation range with a glycerol-based, aqueous mountant that does not maximum at about 535 nm and emission at about solidify but remains a viscous liquid on the slide. 615 nm when bound to DNA. After mounting, coverslipped slides will not readily dry out and can be reviewed for weeks afterwards VECTASHIELD ® Mounting Medium without sealing. For prolonged storage, coverslips H-1000 • 10ml can be permanently sealed around the perimeter VECTASHIELD ® Mounting Medium with nail polish. with DAPI H-1200 • 10ml ® VECTASHIELD ® Hard !Set ™ Mounting Medium is an VECTASHIELD Mounting Medium aqueous mountant that hardens when allowed to with PI H-1300 • 10ml ® ™ set at room temperature for at least 20 minutes. This VECTASHIELD Hard !Set Mounting Medium facilitates handling of the slide, eliminates the need H-1400 • 10ml ® ™ to secure the coverslip, and is more convenient for VECTASHIELD Hard !Set Mounting Medium use with oil immersion microscopy. Although with DAPI H-1500 • 10ml stained and mounted slides may be stored for sev eral weeks, VECTASHIELD ® Hard !Set ™ is not a permanent mounting medium. To remove coverslip and mounting medium, slides can simply be incu - bated in buffer until the mounting medium is re - moved. Slides can be remounted in fresh mounting medium.

Fluorescence in situ hybridization of human chromosomes using FastTag ® Fluorescein-labeled pHuR 98 detected with Biotinylated Anti-Fluorescein and Fluorescein Avidin DCS, and FastTag ® Texas Red ® - labeled pUC1.77 directly detected. Counterstained and mounted with VECTASHIELD ® with DAPI.

www.vectorlabs.com 13

Chromogenic Substrates for Enzyme-based DuoLuX ™ Chemiluminescent/Fluorescent Detection Substrates The reagents in these enzyme substrate kits are For Peroxidase SK-6604 • 200 ml supplied as concentrated stock solutions in conven - For Alkaline Phosphatase SK-6605 • 100 ml ient dropper bottles. All substrates can be used in ISH and blotting applications. DuoLuX ™ Chemiluminescent/ Fluorescent Substrate is Alkaline Phosphatase a unique formula based on acridan chemistry with very high sensitivity, prolonged chemilumi nescent BCIP/NBT Substrate Kit SK-5400 • 1 Kit light emission, as well as permanent flu orescence. ® Vector Red Substrate Kit The DuoLuX ™ Substrate is available for alkaline SK-5100 • 1 Kit phosphatase 39, 53, 55 or peroxidase 10, 17, 43 and is recommended for blotting applications. 16, 25, 48 BCIP/NBT substrate (blue/violet) is the most sensi - Sensitivities ranging from 100 fg to 10 pg of nucleic tive of all the chromogenic substrates because it acid can be achieved. continues to develop over many hours, darkening with time. The UltraSNAP ™ Detection Kit , which includes the ™ Vector ® Red is unique. It is chromogenic and flu o- DuoLuX Substrate, is designed for detection of rescent, and dehydratable. It can be viewed with a bi otinylated probes in nucleic acid blotting applica - fluorescence microscope using a rhodamine or Texas tions (Please see page 14 for details). Red ® filter. Vector ® Red can be used in fluorescent ™ multiple label protocols in combination with fluo - With either substrate formula, reacted DuoLuX rochromes like fluorescein and is compatible with sub strate luminesces in the blue range with a peak fluorescent counterstains like DAPI. Vector ® Red emission at 453 nm. The half-life of luminescent can also be used for sequential, multiple label ISH emission exceeds that of other luminescent sub - or IHC/ISH because of its stability through ISH pro - strates. The fluorescent signal can be seen visually 7, 34 cedures. or captured digitally. The excitation maximum is at 405 nm, but other wavelengths (254 nm and The alkaline phosphatase substrate kits provide 365 nm) also excite. Maximum fluorescent emission sufficient reagents to make approximately 200 ml of occurs at 453 nm. working solution.

Peroxidase Permanent Mounting Medium for Chromogenic ImmPACT ™ DAB SK-4105 • 120ml Substrates DAB Substrate Kit SK-4100 • 1 Kit VectaMount ® Permanent Mounting Medium TMB Substrate Kit SK-4400 • 1 Kit Vector ® NovaRED ™ H-5000 • 60ml Substrate Kit SK-4800 • 1 Kit Vector ® VIP Substrate Kit SK-4600 • 1 Kit VectaMount ® Mounting Medium is a non-flammable, non-hazardous, optically clear, odorless formula Vector ® NovaRED ™ (red), Vector ® VIP (purple), for permanently preserving enzyme substrates in DAB and DAB/Ni (brown or black), are similar in tissue sec tions or cell preparations. VectaMount ® sensitivity and can be permanently mounted. All Mounting Medium retains the color and i ntensity produce dense precipitates with crisp localization. of preparations stained with en zyme substrates ImmPACT ™ DAB has these same attributes but is such as BCIP/NBT, Vector ® Red, DAB, ImmPACT ™ up to four times more sensitive. DAB or ImmPACT ™ DAB, TMB, Vector ® NovaRED ™, and Vector ® VIP. DAB can be used for multiple label ISH or IHC/ISH because of their stability. TMB is the most sensitive peroxidase substrate, albeit with a more diffuse, less lo calized reaction product. Each peroxidase substrate kit provides all reagents necessary to prepare approximately 300 ml of working solution. ImmPACT ™ DAB provides 120 ml of working solution. 14

Applications

Southern and Northern Blot DuoLuX ™ Substrate Chemiluminescence Hy bridization Fluorescence

FastTag ®-, EndTag ™ -, and PHOTOPROBE ® Biotin- AP la beled probes can be used to detect targets on Streptavidin Southern and northern blots as well as colony or plaque lifts. The same well established protocols Biotinylated for radioactive hybridization are employed but probe with out the hazards and disposal issues associated Target DNA with radioactivity.

Hybridization of the nucleic acid probe to the Chemiluminescence can be documented and target on the blot is simplified using the ready- recorded by expo sure to film or by image capture to-use HYBEX ™ Hybridization Solution . HYBEX ™ with digital imaging systems. Blots can be exposed Hy bridization Solution contains no formamide and to film several times over an extended period of can be used with both nylon and nitrocellulose time, with typical film exposure times of 30 seconds membranes. to 10 minutes. Sensitivities as low as 100 fg of tar get nucleic acid can be achieved. Optimal expo - DNA Molecular Weight Markers can be used as sure times using digital imaging systems de pend easy size references. These ladders range from on the sensitivity of the instru ment. Chemilumi - 0.2 to 10 kbps, are available biotinylated or un - nescence can be developed on either nylon or la beled, are ready-to-use, and contain loading dye nitrocellulose membranes, although nylon is for monitoring electrophoretic mobility. pre ferred because of faster sig nal production.

The UltraSNAP ™ Detection System for Nucleic Alternatively, the fluorescence of the developed Acid Blots allows for the convenient detection of substrate can be recorded with a digital imaging biotinylated nucleic acids on nylon or nitrocellu lose system or conventional camera even months after blots. 32, 40, 54 (Please see protocol on page 20). This the chemiluminescence has faded. Acquiring an kit features the ™ Chemiluminescent/ Fluores - DuoLuX image from the fluorescent signal requires a much cent Substrate (see page 13), alkaline phosphatase shorter exposure time than from the chemilumi - streptavidin, and a specially optimized PolyBlock ™ nescent signal, often just a fraction of a second. Blocking Reagent and washing solutions developed for high sensitivity and low background. One kit Selected Reagents: contains sufficient reagents to develop approxi - mately twenty 100 cm 2 blots. HYBEX ™ Hybridization Solution MB-1230 • 200ml Detection is accomplished using biotinylated DNA Molecular Weight Markers probes that are recognized by streptavidin coupled Unlabeled MB-1301 • 25µg to alkaline phosphatase. The probe is visualized Biotinylated MB-1302 • 25µg by the conversion of the ™ Substrate to a DuoLuX 10x Casein Solution SP-5020 • 250ml lu minescent and fluorescent product by a dephos - Animal-Free Blocker ™ (5x) phorylation reaction catalyzed by alkaline phos - SP-5030 • 250ml phatase. The sensitivity is enhanced with the use UltraSNAP ™ Detection Kit of the PolyBlock ™ Blocking Reagent and wash MB-6500 • 1 Kit buffers included in the kit. UltraSNAP ™ Accessory Kit (buffers and blocking agent only) MB-6501 • 1 Kit

www.vectorlabs.com 15

In Situ Hybridization Comparative Genomic Hybridization The availability of many different labels and the wide range of detection systems allows the flexibility Comparative genomic hybridization (CGH), based and sensitivity necessary for in situ hy bridization on fluorescence in situ hybridization, is a technique (ISH). For example, for fluorescence in situ hy - for screening chromosomal imbalances in tumor bridization, biotin-labeled probes can be detected tis sue. Labeled DNA samples from two sources are with fluorescently labeled avidin or streptavidin. competitively hybridized onto normal chromosome For brightfield microscopy, alkaline phosphatase- metaphase spreads: one labeled DNA sample is conjugated streptavidin followed by the enzyme iso lated from tumor tissue, the other, differently substrate, BCIP/NBT, provides a sensitive detection labeled DNA from normal tissue. Based on the method. Additional color choices are available with dif ferential hybridization of the two different ® the use of the peroxidase-based VECTASTAIN sources of DNA, the over- or under-e xpression of ABC Kit followed by the appropriate substrate (e.g. chromosomal regions can be mapped on the normal ™ ® ® TMB, DAB, ImmPACT DAB, Vector VIP, or Vector chromosomes. NovaRED ™). PHOTOPROBE ® Biotin has been shown to be an Haptens such as dinitrophenyl (DNP), fluorescein, ef fective labeling technique of highly degraded ® and Texas Red , for example, provide nearly equally DNA from archival material for this analysis .26, 27, 59 sensitive alternatives to biotin. Several detection Often, DNA isolated from formalin-fixed, paraffin- options are also available for these la bels. Anti - embedded archival tissue is reduced in quality, bodies to these haptens, together with many fluo - tending to be smaller than 1000 bp. Using the rescent and enzymatic detection methods, provide a classical method of nick translation, the resulting great array of possibilities for detecting probes. labeled DNA fragments are usually too small to give a detectable signal. Because l abeling with With the options available, the researcher has PHOTOPROBE ® Biotin is non-de structive, the substantial flexibility in optimizing experimental original, larger fragments of DNA are labeled al - systems, especially in cases where endogenous biotin lowing for the analysis of gains and losses of DNA or enzyme activity, or antibody cross-reac tivity from the tumor tissue sam ple. Over-represented present potential problems. The available options chromosomal regions may correspond to tumor also allow the simultaneous detection of multiple, promoting genes, and chromosomal deletions differentially labeled probes in a single experiment. po tentially contain tumor suppressor genes. (See pages 22-25).

Optimization of Probe Length for In Situ Hybridization

NIC KIT ™ p.s.o. has been developed to reduce the size of large DNA probes using a photo/enzymatic procedure without the problem of over- or under- di gestion experienced with other methods. A smaller probe size generally results in better signal and lower background for ISH applications, probably due to better tissue penetration of the probe. CGH sum karyogram was created after analyzing PHOTOPROBE ® ™ labeled tumor DNA from a small cell lung cancer. Biotin was NIC KIT p.s.o. Probe Size Optimization Kit detected with Vector ® Fluorescein Avidin D (green). Chromosomes MB-1905 • 1 Kit from 15 metaphase spreads were analyzed for each hybridiza - tion. Figure provided courtesy of Dirk Korinth, Konrad Donhuijsen, Ulrike Bockmühl, and Iver Petersen, Institute of Pathology, University Hospital Charité, Berlin, Germany. 16

Cellular Localization EndTag ™-Labeled Primers Used in PCR Amplification FastTag ®-, EndTag ™ -, and PHOTOPROBE ® Biotin- labeled nucleic acids have been successfully employed Several applications involve the incorporation of in the study of intracellular trafficking of gene labeled primers into a PCR amplification product: de livery complexes and nuclear import mechanisms of viral nucleoprotein molecules. Because these • Immobilization of DNA target to a solid support la beling systems do not destroy the nucleic acid for isolating sequence-specific DNA-binding mol e- nor create nascent nucleic acid, the original DNA cules. Synthesizing a DNA fragment by PCR using or RNA can be tagged and traced throughout the a labeled primer ensures that the label is located experiment. only near the end of the DNA. The target DNA is thus bound to the matrix only at one end, minimiz - For example, in the study of gene delivery, plasmid ing interference in the binding domains. DNA can be tagged with a FastTag ® label. Intracel - lular traf ficking of gold-labeled plasmid DNA can • Cloning or library screening by hybrid capture. be fol lowed under electron microscopy. 13, 14 In Immobilized single-stranded DNA can be prepared by addi tion, fluorescent staining patterns of flu ores - PCR amplification in which one of a pair of primers is cently labeled plasmid DNA can be followed under EndTag ™-Biotin labeled. Only a single strand of confocal microscopy. 12, 42, 61 In this manner, cellular the PCR product is biotinylated. After binding the interactions of the plasmid DNA with different gene labeled DNA to a matrix such as VECTREX ® Avidin D, delivery vehicles and with cytoskeletal components the unlabeled strand can be removed, yielding a can be studied. Such investigations can yield in - matrix ready for hybrid capture. sights into gene delivery mechanisms. siRNA sam - ples labeled with FastTag ® can be used as internal controls to evaluate transfection efficiency or cell Covalent Attachment of viability. 49 5’ EndTag ™-Labeled Nucleic In a similar manner, 5’ EndTag ™ labeling of nucleic Acids to Gold acid is also used successfully in the study of cellular trafficking and localization of nucleic acids and Nucleic acids containing sulfur atoms can form co - complexes including viral RNA, 35, 36, 44 ribonucleo - valent bonds with gold. T he phosphoro-thioate protein particles, 60 and oligonucleotide/peptide group of 5’ EndTag ™-la beled RNA has b een em - complexes. 57 ployed for immobilization to a gold sub strate for atomic force microscopy. 33

Cellular uptake of labeled plasmid: FastTag ® Fluorescein labeled plasmid DNA (green) incubated with COS-7 cells. Nuclei were counterstained and mounted with VECTASHIELD ® with DAPI (blue).

www.vectorlabs.com 17

Affinity Binding Supernatant Eluate

Fucosylated Genomic/cDNA subtraction, 1, 31, 51 microsatellite –– ++––++ iso lation, 2, 21, 50, 62 and library screening by hybrid VECTREX ® AAL –+ –+ – +–+ capture 24 require the removal or immobilization of nucleic acid hybrids. Affinity binding matrices are available for use with PHOTOPROBE ® -, FastTag ®-, or EndTag ™- labeled probes for such applications.

Irreversible binding of biotinylated nucleic acids ® Reversible binding of FastTag Fucose-labeled λ Hin d III DNA to to a solid support can be accomplished using VECTREX ® AAL. Labeled (+) or unlabeled (-) DNA was incubated ® ® with VECTREX AAL (+) or binding buffer (-). Following centrifu - VECTREX Avidin D. Following incubation of gation of binding reactions, VECTREX ® AAL was washed and the VECTREX ® Avidin D/biotinylated probe with a bound DNA eluted with L-fucose . Supernatants and eluates from cellular extract, hybridized nucleic acids can be the binding reactions were fractionated by agarose gel elec - trophoresis and DNA visualized by ethidium bromide staining. Lane recov ered by melting the doubled stranded nucleic 4 shows that the fucosylated DNA bound to the VECTREX ® AAL and acid and eluting the unbiotinylated single strands. 4, lane 8 shows that fucosylated DNA can be easily eluted. 23, 41 (Guidelines for this procedure are described on page 26). Irreversible Binding Matrix VECTREX ® Avidin D A-2020 • 1 ml Reversible binding of nucleic acids can be achieved with either of two systems: Reversible Binding Matrices 1. VECTREX ® Avidin DLA matrix has a significantly reduced binding affinity for biotin. Biotinylated For Biotin Labeled Nucleic Acids nucleic acids that are bound to this matrix can be VECTREX ® Avidin DLA MB-2021 • 1 ml eluted with biotin. 8 VECTREX ® Avidin DLA 2. Nucleic acids that have been labeled with Binding and Elution Kit MB-2022 • 1 Kit FastTag ® Fucose or EndTag ™ Fucose bind specifically to VECTREX ® AAL, a matrix containing the fucose- specific lectin from Aleuria aurantia . These nucleic For Fucose Labeled Nucleic Acids acids are then easily eluted under mild conditions ® using the sugar L-fucose at physiological pH and VECTREX AAL MB-1396 • 1 ml salt concentrations. VECTREX ® AAL Binding and Elution Kit MB-1397 • 1 Kit 18

Resolve Same-Sized DNA by Amplification of Fluorescent Sequence Variation Signal in Nucleic Acid Microarrays

The ability to resolve DNAs of similar size in a gel Fluorescent amplification of biotinylated probes is is critical for applications such as differential display common in microarray-based analysis. The multiple and Rapid Amplification of Polymorphic DNAs binding capability of Biotinylated Anti-Streptavidin (RAPD) in which multiple DNA species need to be provides a method for significant signal amplifica - separated before subsequent excision and analysis. tion. This antibody binds to streptavidin through the Two sequence specific DNA ligands in the Resolve-It ™ antigen binding sites and through the covalently Kit bind to GC- or AT- rich sites on DNA and retard attached biotin residues. Following the first appli - the electrophoretic migration of DNA in a sequence cation of a fluorescent streptavidin such as phyco - specific manner. AT-Yellow ™, a minor groove bind - erythrin- or fluorescein-labeled streptavidin, the ing bisbenzimide-PEG conju gate, binds to AT-rich signal is am plified by incubation with Biotinylated regions; GC-Red ™, an intercalating phenyl neutral Anti-Streptavidin followed by a second incubation red-PEG conjugate, binds GC-regions. A ligand is with the fluorescent streptavidin. The same pro - added to the agarose during gel preparation, and, cedure can be performed with labeled avidin and during electrophoresis, the increased friction Biotinylated Anti-Avidin. This procedure results caused by ligand association with the DNA retards in the introduction of more fluo rochromes at the the mobility of DNA depending on the amount of target site. 3, 6, 9, 38 ligand bound. The resolved bands can be excised and electrophoresed on a gel containing the second Biotinylated Anti-Streptavidin ligand, if desired, for confirmation of homogeneity made in goat BA-0500 • 0.5mg or for additional resolution. The resolved DNA Phycoerythrin Streptavidin sequences can then be extracted for cloning, SA-5007 • 1mg sequencing, or other analysis. The quantity of Fluorescein Streptavidin SA-5001 • 1mg each ligand supplied is sufficient to prepare about 200 ml of agarose gel.

Resolve-It ™ Kit - Sequence Specific DNA Ligands Amplification of Signal Using Biotinylated MB-1401 • 1 Kit Anti-Streptavidin Biotinylated Target

Step 1: Add Fluorescent Streptavidin

Step 2: Add Biotinylated A B Anti-Streptavidin

Two 600 bp DNAs of different sequence form a single band using typical electrophoretic conditions (A). The same DNAs are separated into two distinct bands in a gel containing Resolve-It ™ AT-Yellow ™ (B). Step 3: Add Fluorescent Streptavidin

www.vectorlabs.com 19

Protocols

Assessing Labeling Efficiency Quantitation of Biotin using the Quant *Tag ™ Biotin Kit In applications involving several steps - from produc - ing and labeling a probe to detecting the labeled For biotin-labeled probes, labeling efficiency can probe - assessing labeling efficiency can be an be determined using either the dot blot technique es sential part of assay design. or an alternative method - Quant *Tag ™ Biotin quantitation. The Quant *Tag ™ Biotin Kit is designed Estimation of Labeling by to determine the amount of free bi otin in solution Comparative Dot Blot or the number of biotins attached to nucleic acids, proteins, or other macromolecules. The kit The efficiency of labeling can be estimated by reagents chemically react with free or bound biotin, com paring the relative detection sensitivity of t he producing a colored product that can be quantified labeled nucleic acid to a standardized sam ple in a using a spectrophotometer. The absorbance is side by side dot blot dilution series. measured in the visible spectrum, allowing the use of plastic cuvettes or microtitre plates. 1. Dilute both the labeled nucleic acid sample and the labeled DNA standard to 1 µg/ml, 100 ng/ml, 10 Unlike other methods of measuring biotin incor - ng/ml, and 1 ng/ml in TE. poration, no pre-digestion of the nucleic acid is re quired, saving time and increasing consistency. 2. Dot 1 µl of each dilution on nitrocellulose or The assay can be completed in 30 minutes, and is nylon membrane. Crosslink the membrane accord - able to detect less than 1 nmol of biotin. ing to the manufacturers’ protocol. Quant *Tag ™ Biotin Kit BDK-2000 • 1 Kit 3. Block the membrane in blocking solution

4. Detect by incubating with the appropriate alkaline phosphatase conjugate and an AP substrate such Biotin Quantitation using Quant Tag ™ Biotin Kit as BCIP/NBT or DuoLuX ™ Chemiluminescent/Fluores - * cent Substrate according to the instructions pro - vided with the substrate. Efficiency of labeling can be estimated by the comparison of the signals of the labeled sample and the labeled DNA standard.

Selected Reagents:

10x Casein Solution SP-5020 • 250ml Animal-Free Blocker ™ SP-5030 • 250ml BCIP/NBT Substrate Kit SK-5400 • 1 kit DuoLuX ™ Chemiluminescent/Fluorescent Substrate for Alkaline Phosphatase SK-6605 • 100 ml

The sample to be tested is reacted with the kit reagents along with standards containing known amounts of biotin. The ab - sorbance readings of the known samples are plotted producing a standard curve. The absorbance of the test sample is located on the standard curve indicating the amount of biotin present. 20

Protocol: Southern Blot Hybridization and Detection Perform Southern transfer and hybridization of the Detection : This protocol is adapted from the Ultra - biotinylated probe followed by stringency washes SNAP ™ Detection Kit which has been optimized for using standard procedures. 46 the chemilumines cent or fluorescent detection of biotinylated nu cleic acid. Solution volumes are for Blotting can be done onto either nylon or nitro - a 100 cm 2 blot and can be scaled accordingly. A cellulose (nylon membranes usually re quire a general protocol using the chromogenic alkaline shorter exposure time than nitrocellulose and are phosphatase substrate, BCIP/NBT, as the substrate therefore recommended, especially when using is described on the following page. CCD camera-based digital imagers). 1. Block blot in 20 ml of 1x PolyBlock ™ Reagent Hybridization : Prehybridization and hybridization for 30 minutes at room temperature with gentle incubations can be carried out in appropriate hy - shaking. bridization tubes or in heat-sealable polyethylene bags. To ensure constant agitation, incubations can 2. Incubate the blot in diluted Alkaline Phosphatase- be performed in a commercial hybridization oven Streptavidin (1 µg/ml in 1x PolyBlock ™ Reagent) or water bath equipped with a shaking platform. for 30 minutes at room temperature with gentle shaking. Prehybridization : Preincubate the membrane in sufficient HYBEX ™ Hybridization Solution to ensure 3. Wash blot 3 times for 10 minutes each in 30 ml that the membrane is completely covered (usually of 1x Wash A at room temperature with gentle 0.2 ml of hybridization solution is enough for each shaking. square centimeter of membrane). Nitrocellulose membranes are prehybridized for 1-2 hours and 4. Rinse blot in 30 ml of 1x Wash B. nylon membranes for 30 minutes. Prehybridization temperature should be the same as the temperature 5. Remove excess Wash B by touching edge of blot used for hybridization. to absorbent paper.

Hybridization : Double stranded DNA probes need 6. Place blot target-side up on plastic wrap on a to be denatured before hybridization. Dilute dena - level surface. tured probe in HYBEX TM solution. Use sufficient hy bridization solution to ensure that the membrane 7. Cover the blot surface with DuoLuX ™ Substrate will be completely covered. (about 5 ml). Incubate for 5 minutes under subdued light or in the dark. Decant prehybridization solution and immediately add diluted probe to the membrane. Optimal 8. Wash the blot in 30 ml of 1x Wash B for 1 hybridization temperature will depend on the minute at room temperature with gentle shaking. probe type and size and may have to be empiri - Remove excess liquid from the blot by touching cally determined. As a guideline, temperatures can edge to absorbent paper. See Note A . range from 68 °C for large DNA fragments to room temperature for short oligonucleotide probes. (For 9. Place the blot in a sheet protector or between oligonucleotide probes it is necessary to determine layers of plastic wrap and smooth away any bubbles the melting temperature of the oligo). trapped between layers. The signal can be recorded with either film or a digital imager. Incubate the membrane with the HYBEX ™ solution containing the probe for two hours to overnight. 10a. Chemiluminescence : Following hybridization, wash the membrane with stringency solutions: 2 x 5 min in 2x SSC, 0.1% SDS, X-ray film: Expose to film for 5 seconds to several followed by 2 x 15 min in 0.1x SSC, 0.1%SDS. (1x SSC hours. See Note B . is 0.15 M NaCl, 15 mM Trisodium Citrate, pH 7.0). Digital imager: For optimal exposure time, please consult the manufacturer of the digital imaging system. A digital imager also enables recording of fluorescent signal. See Notes C and D .

www.vectorlabs.com 21

10b. Fluorescence: General Protocol for Southern Blot Detection Using BCIP/NBT Substrate for DuoLuX ™ fluorescence signal can be achieved with Visualization a UV transilluminator. The signal can be ac quired with a digital imager or conventional camera, 1. Block blot in 20 ml of 1x Casein Solution for 30 equipped with an ethidium bromide fil ter. Pre- minutes at room temperature with gentle shaking. exposure to UV light for at least 2 minutes fre - quently enhances the fluorescent sig nal. DuoLuX ™ 2. Incubate the blot in diluted Alkaline Phosphatase- fluorescent signal can usually be captured in just a Streptavidin (1 µg/ml in 1x Casein Solution) for 30 few seconds. However, for the best picture quality minutes at room temperature with gentle shaking. some optimization may be needed. The fluores - cence of the developed sub strate can be recorded 3. Wash blot 3 times for 5 minutes each in 30 ml of months after the chemiluminescence has faded. TBST (Tris buffered saline, pH 7.5, with 0.1% See Note D . Tween) at room temperature with gentle shaking.

Notes: 4. Rinse blot for 5 minutes in 30 ml 0.1 M Tris, pH A. Extensive washing will reduce signal strength; do 9.5. not extend the wash time unless high background is ex peri enced. If background is excessive, repeat steps 6-9 5. Prepare BCIP/NBT substrate solution according with a wash time of 5-10 minutes in step 8. Ideal wash time to kit instructions in a separate staining dish, and is dependent on the degree of background previously submerge the blot in the solution until signal de - de tected and, therefore, may require optimization. velops.

B. The long emission lifetime of DuoLuX ™ allows the user Probes other than biotin can be detected by substi - to re-expose the same blot until optimal signal to noise tuting Alkaline Phosphatase-Streptavidin in step 2 ratio is achieved. Typical exposure times are from 5 to 60 with the appropriate AP-conjugated antibody. minutes. Selected Reagents: C. Exposure time for chemiluminescent signal development will depend on the sensitivity of the instrument. Often, HYBEX ™ Hybridization Solution imagers require longer exposure times than x-ray film. MB-1230 • 200ml DNA Molecular Weight Markers D. Prolonged exposure to UV will weaken chemilumines - Unlabeled MB-1301 • 25µg cent emission. If fluorescence imaging is to be performed, Biotinylated MB-1302 • 25µg chemiluminescence should be recorded first. 10x Casein Solution SP-5020 • 250ml Animal-Free Blocker ™ (5x) SP-5030 • 250ml BCIP/NBT Substrate Kit SK-5400 • 1 Kit UltraSNAP ™ Detection Kit MB-6500 • 1 Kit UltraSNAP ™ Accessory Kit* MB-6501 • 1 Kit

*Contains PolyBlock ™ Reagent and wash buffers only 22

Protocol: Fluorescent Detection of Biotin-Labeled ISH Probes

This procedure uses successive rounds of Fluorescein 8. Wash slides 2 x 5 minutes in 4x SSC + 0.1% Tween Avidin DCS and Biotinylated Anti-Avidin to detect 20 before coverslipping with a VECTASHIELD ® and amplify in situ hybridization signals. The multiple Mounting Medium. binding capability of Biotinylated Anti-Avidin D can provide significant amplification. This antibody * Note: 5% nonfat dry milk plus 0.1% Tween 20 in 4x SSC binds to Avidin D through the antigen binding sites can be used as an alternative blocking solution. (4x SSC is and through the biotin residues that are covalently 0.6 M NaCl, 60 mM sodium citrate, pH 7.0.) However, attached to the molecule. Following the first non-fat dry milk can contain variable amounts of biotin ap plication of Fluorescein Avidin DCS, the signal is which could reduce staining if used as a diluent for amplified by incubation with Biotinylated Anti-Avidin, (strept)avidin conjugates. followed by a second incubation with Fluorescein Avidin DCS. This results in the introduction of Detection Reagents sev eral more fluorochromes at the target site. The same procedure can be performed with labeled ISH Blocking Solution (5x) streptavidin and Biotinylated Anti-Streptavidin MB-1220 • 100ml (see diagram on page 18). Fluorescein Avidin DCS A-2011 • 1mg 1. After hybridization of biotinylated DNA/ RNA Biotinylated Anti-Avidin probes, block tissue sections or chromosome made in goat BA-0300 • 0.5mg spreads for at least 30 minutes in 1x ISH Blocking Biotinylated Anti-Streptavidin Solution*. The effectiveness of the blocking solution made in goat BA-0500 • 0.5mg may be enhanced by pre-warming the solution to 37 ºC and incubating tissue sections or chromosome spreads for 30 minutes or longer at 37 ºC. Mounting Media

2. Dilute each of the detection reagents (Fluorescein VECTASHIELD ® Mounting Medium Avidin DCS and Biotinylated Anti-Avidin; or fluores - H-1000 • 10ml cent streptavidin and Biotinylated Anti-Streptavidin) VECTASHIELD ® Mounting Medium to 5 µg/ml in 1x ISH Blocking Solution approximately with DAPI H-1200 • 10ml 30 minutes before use to minimize any non-specific VECTASHIELD ® Mounting Medium binding. with PI H-1300 • 10ml VECTASHIELD ® Hard !Set ™ Mounting Medium 3. Drain off the blocking solution from the specimen H-1400 • 10ml and add the Fluorescein Avidin DCS solution (5 µg/ml). VECTASHIELD ® Hard !Set ™ Mounting Medium Incubate for 30 minutes at room temperature. with DAPI H-1500 • 10ml

4. Wash slides for 2 x 3 minutes in blocking solution.

[If satisfactory sensitivity has been achieved, skip to step 8. To amplify the fluorescent signal, continue with steps 5 through 7].

5. Incubate sections or spreads with the Biotiny - lated Anti-Avidin solution (5µg/ml) for 30 minutes at room temperature.

6. Wash slides for 2 x 3 minutes in blocking solution.

7. Perform a second incubation of the sections or spreads with the same Fluorescein Avidin DCS so lution (5 µg/ml) for 30 minutes at room temper - ature.

www.vectorlabs.com 23

Protocol: Fluorescence Detection of Fluorescein-Labeled ISH Probes

2. Dilute each of the detection reagents (Biotiny - Mounting Media lated Anti-Fluorescein and Fluorescein Avidin DCS) to 10 µg/ml in 1x ISH Blocking Solution for approxi - VECTASHIELD ® Mounting Medium mately 30 minutes before use to minimize any non- H-1000 • 10ml specific binding. VECTASHIELD ® Mounting Medium with DAPI H-1200 • 10ml 3. Drain off the blocking solution from the specimen VECTASHIELD ® Mounting Medium and incubate with Biotinylated Anti-Fluorescein solu - with PI H-1300 • 10ml tion (10 µg/ml) for 30 minutes at room temperature. VECTASHIELD ® Hard !Set ™ Mounting Medium H-1400 • 10ml 4. Wash slides for 2 x 3 minutes in blocking solution. VECTASHIELD ® Hard !Set ™ Mounting Medium with DAPI H-1500 • 10ml 5. Incubate with the Fluorescein Avidin DCS solution (10 µg/ml) for 30 minutes at room temperature.

6. Wash slides 2 x 5 minutes in 4x SSC + 0.1% Tween 20 before coverslipping with a VECTASHIELD ® Mounting Medium.

* Note: 5% nonfat dry milk plus 0.1% Tween 20 in 4x SSC can be used as an alternative blocking solution. (4x SSC i s 0.6 M NaCl, 60 mM sodium citrate, pH 7.0.) However, non-fat dry milk can contain variable amounts of biotin which could reduce staining if used as a diluent for (strept)avidin conjugates. 24

Protocol: Chromogenic Detection of Biotin-Labeled ISH Probes

1. After hybridization of biotin-labeled DNA/RNA Detection Reagents probes, block tissue sections or chromosome spreads for at least 30 minutes in 1x ISH Blocking ISH Blocking Solution (5x) Solution. The effectiveness of the blocking solution MB-1220 • 100ml may be enhanced by pre-warming the solution to Alkaline Phosphatase Streptavidin 37 ºC and incubating tissue sections or chromosome SA-5100 • 1ml spreads for 30 minutes or longer at 37 ºC. Please BCIP/NBT Substrate Kit SK-5400 • 1 kit see Note A . Levamisole Solution SP-5000 • 18ml Vector ® Nuclear Fast Red Counterstain 2. Dilute Alkaline Phosphatase Streptavidin to H-3403 • 500ml 1–5 µg/ml in 1x ISH Blocking Solution approxi - Vector ® Methyl Green Counterstain mately 30 minutes before use to minimize any H-3402 • 500ml non-specific binding. Please see Note B . Mounting Media 3. Drain off the blocking solution from the specimen and incubate with Alkaline Phosphatase Strepta - VectaMount ™ Permanent vidin solution for 30 minutes at room temperature. Mounting Medium H-5000 • 60ml VectaMount ™ AQ Aqueous 4. Wash slides for 2 x 3 minutes in 100 mM Tris, pH Mounting Medium H-5501 • 60ml 9.5 buffer.

5. Visualize the probe by incubating the tissue section or chromosome spread in BCIP/NBT sub - strate working solution prepared according to kit instructions (BCIP/NBT Substrate Kit). Incubate until desired sensitivity is achieved.

6. Wash in 100 mM Tris, pH 9.5 buffer for 5 minutes.

7. Rinse in tap water and counterstain if desired (BCIP/NBT substrate is compatible with Vector ® Nu clear Fast Red counterstain and Vector ® Methyl Green counterstain).

8. For permanent mounting, dehydrate, clear, and mount sections in VectaMount ™ Mounting Medium which minimizes crystal formation in mounted sec - tions. For aqueous mounting, use VectaMount ™ AQ Mounting Medium.

Notes: A. 5% nonfat dry milk plus 0.1% Tween 20 in 4x SSC can be used as an alternative blocking solution. (4x SSC i s 0.6 M NaCl, 60 mM sodium citrate, pH 7.0.) However, non-fat dry milk can contain variable amounts of biotin which could reduce staining if used as a diluent for (strept)avidin conjugates.

B. For an overnight incubation in the BCIP/NBT substrate solution, use the Alkaline Phosphatase Streptavidin reagent at a concentration of 0.3 – 0.5 µg/ml.

www.vectorlabs.com 25

Protocol: Chromogenic Detection of Fluorescein-Labeled ISH Probes

1. After hybridization of fluorescein-labeled Detection Reagents DNA/RNA probes, block tissue sections or chromo - some spreads for at least 30 minutes in 1x ISH ISH Blocking Solution (5x) Blocking Solution. The effectiveness of the blocking MB-1220 • 100ml solution may be enhanced by pre-warming the Alkaline Phosphatase Anti-Fluorescein so lution to 37 ºC and incubating tissue sections or MB-2100 • 150µg chromosome spreads for 30 minutes or longer at BCIP/NBT Substrate Kit SK-5400 • 1 kit 37 ºC. Please see Note A . Levamisole Solution SP-5000 • 18ml Vector ® Nuclear Fast Red Counterstain 2. Dilute Alkaline Phosphatase Anti-Fluorescein to H-3403 • 500ml 5 µg/ml in 1x blocking solution approximately 30 Vector ® Methyl Green Counterstain minutes before use to minimize any non-specific H-3402 • 500ml binding. Please see Note B . Mounting Media 3. Drain the blocking solution from the specimen and incubate with Alkaline Phosphatase Anti- VectaMount ™ Permanent Fluorescein solution for 30 minutes at room tem - Mounting Medium H-5000 • 60ml perature. VectaMount ™ AQ Aqueous Mounting Medium H-5501 • 60ml 4. Wash slides for 2 x 3 minutes in 100 mM Tris, pH 9.5 buffer.

5. Visualize the probe by incubating the tissue sec - tion or chromosome spread in BCIP/NBT substrate working solution prepared according to kit instruc - tions. Incubate until desired sensitivity is achieved.

6. Wash in 100 mM Tris, pH 9.5 buffer for 5 minutes.

7. Rinse in tap water and counterstain if desired (BCIP/NBT substrate is compatible with Vector ® Nu clear Fast Red counterstain and Vector ® Methyl Green counterstain).

B. For an overnight incubation in the BCIP/NBT substrate solution, use the Alkaline Phosphatase Anti-Fluorescein reagent at a concentration of 0.2 – 2.0 µg/ml. 26

Protocol: Methods used to elute single strands of DNA from VECTREX ® - bound biotinylated DNA The procedures outlined below use heat to denature Cited References: the DNA without disrupting the biotin-avidin int er - action. The temperature of washes and elution Carrieo F, Fontanazza G, Cellini F, Giorio G. 2002. will depend on the length and base com position Identification of simple sequence repeats (SSRs) in of the DNA. olive ( Olea europaia L.). Theor Appl Genet. 104:301-07. According to Carriero (2002), PCR amplified genomic DNA was denatured and mixed with biotinylated Kandpal RP, Kandpal G, and Weissman SM. 1994.

oligonucleotide (GA) 20 . This mixture was allowed Construction of libraries enriched for sequence to anneal and was mixed with VECTREX ® Avidin D. repeats and jumping clones, and hybridization After two washes to remove unbound DNA, a se lection for region-specific markers. PNAS. more stringent wash at 55 °C was employed. 91:88-92. Single- stranded DNA was collected by incubating in solution at 65 °C. Rehner SA and Buckley EP. 2003. Isolation and characterization of microsatellite loci from the Kandpal (1994) heat denatured a genomic digest entomopathogenic fungus Beauferia bassiana or PCR product and hybridized it to a biotinylated (Ascomycota: Hypocreales). Mol Ecol Notes. 29-base oligomer. The hybridization mixture was 3:409-11. added to VECTREX ® Avidin D that had been blocked with salmon DNA in 0.1 M Tris, pH 7.5, 150 mM NaCl. The VECTREX ® Avidin D was washed in Tris/HCl to remove unbound salmon DNA. After binding at room temperature, the supernatant was removed. The matrix was washed three times with Tris/NaCl at room temperature, followed by one wash with the same buffer at 50°C. Single-stranded DNA was recovered by incubation at various stringencies.

(AGAT) 11 repeats were dissociated in 150 mM NaCl at 65 °C. (CAG) 15 repeats were recovered in 15 mM NaCl at 65 °C, while (CA) n repeats were dissociated in water at 65 °C.

Rehner (2003) annealed PCR products to biotiny - lated oligonucleotides specifying CA, GA, AAG, or GTT motifs. These hybrids were incubated with VECTREX ® Avidin D. After washing in 0.2x SSC/0.1% SDS (1x SSC is 15 mM Citrate, 150 mM NaCl, pH 7.2) to remove unbound DNA, all were suspended in TLE (10 mM Tris, 0.1 mM EDTA, pH 8.0) and heated to 95 °C for 5 minutes to elute sin gle strands (regardless of the biotinylated oligonucleotide used).

www.vectorlabs.com 27

Protocol: Nucleic Acid Purification

There are many methods used to purify nucleic Gel filtration acid samples at different steps in sample prepara - tion (isolation, labeling, etc.). Which is the best Due to the difference in molecular weight, a nucleic suited will depend on the nature of the sample acid sample can be separated from small contami - (DNA, RNA, oligos), the nature of the contaminant nants such as nucleotides, unincorporated labels, (proteins, nucleotides, reactive chemicals) or the small primers, etc., using gel filtration on spin desired level of purity. The simple protocols below columns. For this method, use a commercially can be used for preparation of the samples used in avail able spin column or prepare a spin column protocols on pages 19-26. with Sephadex ® G-25 or other gel filtration matrix with a similar separation range (1 - 3 ml column Ethanol precipitation volume). While the small molecular weight con - taminants enter pores of the gel filtration media, Ethanol precipitation is a commonly used method the large molecular weight sample remains in the to precipitate nucleic acids out of solution due to void volume and is easily recovered by subsequent their low solubility in alcohols. DNA and RNA will centrifugation. precipitate out, leaving soluble contaminants in the solution. 1. Wash the column three times with TEN buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl) by 1. Adjust sample volume to 50 µl with water or TE centrifuging the buffer through the column for 1-2 buffer (10 mM Tris, pH 8.0, 1 mM EDTA). minutes at 1,000 x g (ca. 4,000 rpm). Do not exceed the manufacturer’s recommendations. 2. Add, in this order, the following components: a) 10 µl of 10 M ammonium acetate 2. Load the sample (in TEN buffer) onto the column ≤ b) 2 µl of 1 M MgCl 2 (for nucleic acids 500 bases) and allow to equilibrate for 5 minutes. c) 1 µl of 20 mg/ml glycogen (only necessary with oligonucleotides and dilute nucleic acid solu - 3. Centrifuge the sample (1-2 minutes at 1,000 x g) tions, e.g. ≤10 ng/µl) through the column into a clean tube. d) 125 µl of 95% ethanol (-20 °C)

3. Pellet the precipitated nucleic acid by centrifu ga - tion at 10,000 x g (ca. 13,000 rpm) for 15 minutes.

4. Remove supernatant and wash the pellet with 70% ethanol. Centrifuge at 10,000 x g for 5 minutes.

5. Remove supernatant and resuspend the pelleted nucleic acid in TE. Store at -20 °C to -80 °C for up to 1 year. 28

Fast Facts

Formula for Calculation of Primer Melting Temperature (T m)

Tm (°C) = 81.5 + 16.6(logM) + 0.41(%GC) - (500/n)

n = length of primer M = molarity of the salt in the buffer

Formula for Calculation of nmoles of 5’ or 3’ ends per µl of nucleic acid sample

nmoles ends = A x 1000 nmoles/ µmol BxC

A = the concentration of nucleic acid ( µg/ µl) B = average molecular weight of nucleotide (333 µg/ µmol for DNA; 317 µg/ µmol for RNA) C = total number of bases (for ssDNA or RNA) or base pairs (for dsDNA)

Characteristics of Common Proteins

Protein Molecular Weight Subunits pI Extinction coefficient Optimal pH at 280 nm (at 1 mg/ml) (for enzymes) Alkaline Phosphatase, (calf intestinal) 140,000 2 5.7 1.0 9.5 Avidin 68,000 4 10 1.54 - BSA 67,000 1 4.9 0.67 - GFP 25,000 1 5.0 1.05 - Glucose Oxidase 160,000 2 4.2 1.38 5.5 Horseradish Peroxidase 40,000 1 7.2 0.63 7.0 Immunoglobulin G (IgG) 150,000 4 5-12 1.34 - Streptavidin 60,000 4 5.5 3.4 -

Spectrophotometric Conversions of Nucleic Acids Weight Conversions

1 A 260 of dsDNA = 50 µg/ml = 0.15 mM (in nucleotides) 1 mg = 10 -3 gm 1 A 260 of ssDNA = 33 µg/ml = 0.1 mM (in nucleotides) 1 µg = 10 -6 gm 1 A 260 of ssRNA = 40 µg/ml = 0.11 mM (in nucleotides) 1 ng = 10 -9 gm 1 pg = 10 -12 gm MW average of a deoxyribonucleotide = 333 g/mol 1 fg = 10 -15 gm MW average of a ribonucleotide = 350 g/mol 1 ag = 10 -18 gm

Amino Acid Symbols:

A Alanine G Glycine P Proline R Arginine H Histidine S Serine N Asparagine I Isoleucine T Threonine D Aspartic Acid L Leucine W Tryptophan C Cysteine K Lysine Y Tyrosine Q Glutamine M Methionine V Valine E Glutamic Acid F Phenylalanine

www.vectorlabs.com 29

References

1. Agarwal N et al. 1991. Sequence analysis, expression and 14. Grosse S, Thevenot G, Monsigny M, Fajac I. 2006. Which chromosomal localization of a gene, isolated from a subtracted mechanism for nuclear import of plasmid DNA complexed with human retina cDNA library, that encodes an insulin-like growth polyethylenimine derivatives? J Gene Medicine 8(7):845-51. factor binding protein (IGFBP2). Exp Eye Res. 52:549-61. 15. Gruber HE, Ingram JA, Leslie K, Norton HJ, Hanley EN. 2. An J, Sommer JA, Shore GD, Williamson JE, Brenneman RA, 2003. Cell shape and gene expression in human intervertebral Louis EE. 2004. Characterization of 20 microsatellite marker disc cells: in vitro tissue engineering studies. Biotechnic and loci in the west Indian rock iguana ( Cyclura nubile ). Conserva - Histochem. 78(2):109-17. tion Genetics. 5:121-25. 16. Hall MO, Obin MS, Heeb MJ, Burgess BL, Abrams TA. 3. Black EP, Hallstrom T, Dressman HK, West M, Nevins JR. 2005. Both protein S and Gas6 stimulate outer segment 2005. Distinctions in the specificity of E2F function revealed phagocytosis by cultured rat retinal pigment epithelial cells. by gene expression signatures. Proc Nat Acad Sci USA. Exp Eye Res. 81(5):581-91. 102(44):15948-53. 17. Haruyama T, Sakai T, Matsuno K. 2005. Protein layer 4. Carriero F, Fontanazza G, Cellini F, Giorio G. 2002. coating method on metal surface by electrochemical process Identi fication of simple sequence repeats (SSRs) in olive ( Olea through genetical introduced tag. Biomaterials. 26(24):4944-7. europaia L.). Theor Appl Genet. 104:301-07. 18. Harvard C, Malenfant P, Koochek M, Creighton S, 5. Choi HW, Lemaux PG, Cho M-J. 2002. Use of fluorescence Mickelson ECR, Holden JJA, Lewis MES, Rajcan-Separovic E. in situ hybridization for gross mapping of transgenes and 2005. A variant Cri du Chat phenotype and autism spectrum screening for homozygous plants in transgenic barley disorder in a subject with de novo cryptic microdeletions in - (Hordeum vulgare L.). Theor Appl Genet. 106:92-100. volving 5p15.2 and 3p24.3-25 detected using whole genomic array CGH. Clin Genet. 67:341-51. 6. Choy JS, Tobe BTD, Huh JH, Kron SJ. 2001. Yng2g-depen - dent NuA4 histone H4 acetylation activity is required for mitotic 19. Hiromura M, Suizu F, Narita M, Kinowaki K, Noguchi M. and meiotic progression. J Biol Chem. 276(47):43653-62. 2006. Identification of NBRE (nerve growth factor responsive element) of the TCL1 promoter as a novel negative regulatory 7. Covi JA, Hand SC. 2005. V-ATPase expression during de - element. J Biol. Chem. 281:27753-64. velopment of Artemia franciscana embryos: potential role for proton gradients in anoxia signaling. J Exp Biol. 208:2783-98. 20. Hirota K, Nodasaka Y, Orikasa Y, Okuyama H, Yumoto I. 2005. Shewanella pneumatophori sp. nov., an eicosapen - 8. Cruz P, Ibarra AM, Fiore-Amaral G, Galindo-Sanchez CE, taenoic acid-producing marine bacterium isolated from the Mendoza-Carrion G. 2005. Isolation of microsatellite loci in intestines of Pacific mackerel ( Pneumatophorus japonicus ). green abalone ( Haliotis fulgens ) and cross-species amplification Int J of Systematic Evol Microbiol. 55:2355-9. in two other North American red ( Haliotis rufescens ) and pink (Haliotis corrugata ) abalones. Mol Ecol Notes 5:857-59. 21. Jung J, Lee I, Kim W. 2006. Isolation and characterization of polymorphic microsatellite markers of Anopheles sinensis , a 9. Derocq J-M, Jbilo O, Bouaboula M, Segui M, Clere C, malaria vector mosquito in the East Asia region. Mol Ecol Casel las P. 2000. Genomic and functional changes induced Notes 6(4):1272-74. by the activation of the peripheral cannabinoid receptor CB2 in the promyelocytic cells HL-60. J Biol Chem. 275(21):15621-28. 22. Kandasamy K, Joseph K, Subramaniam K, Raymond JR, Tholanikunnel BG. 2005. Translational control of β2-adrenergic 10. Duncan MK, Kozmik Z, Cveklova K, Piatigorsky J, Cvekl A. receptor mRNA by T-cell-restricted intracellular antigen-related 2000. Overexpression of PAX6(5a) in lens fiber cells results in protein. J Biol Chem. 280(3):1931-43. cataract and upregulation of (alpha)5(beta)1 integrin expression. J Cell Sci. 113:3173-85. 23. Kandpal RP, Kandpal G, Weissman SM. 1994. Construction of libraries enriched for sequence repeats and jumping clones, 11. Fule T, Mathe M, Suba Z, Csapo Z, Szarvas T, Tatrai P, and hybridization selection for region-specific markers. Proc Paku S, Kovalszky I. 2006. The presence of human papillo - Natl Acad Sci USA. 91(1):88-92. mavirus 16 in neural structures and vascular endothelial cells. Virology. 348:289-96. 24. Kandpal RP, Ward DC, Weissman SM. 1990. Selective enrichment of a large size genomic DNA fragment by affinity 12. Funhoff AM, van Nostrum CF, Lok MC, Kruijtzer JAW, capture: an approach for genome mapping. Nucleic Acids Res. Crommelin DFA, Hennink WE. 2005. Cationic polymethacry - 18:1789-95. lates with covalently linked membrane destabilizing peptides as gene delivery vectors. J Controlled Release. 101:233-46. 25. Knezevic V, Leethanakul C, Bichsel VE, Worth JM, Prabhu VV, Gutkind JS, Liotta LA, Munson PJ, Petricoin EF, Krizman 13. Grosse S, Aron Y, Thevenot G, Francois D, Monsigny M, DB. 2001. Proteomic profiling of the cancer microenvironment Fajac I. 2005. Potocytosis and cellular exit of complexes as by antibody arrays. Proteomics. 1(10):1271-78. cellular pathways for gene delivery by polycations. J Gene Med. 7:1275-86. 30

26. Korinth D, Donhuijsen K, Bockmuhl U, Petersen I. 2004. 39. Puri V, Puri S, Svojanovsky SR, Mathur S, Macgregor RR, PHOTOPROBE ® biotin: An alternative method for labeling Klein RM, Welch KMA, Rerman NEJ. 2006. Effects of estrogen archival DNA for comparative genomic hybridization. Cell on trigeminal ganglia in culture: implications for hormonal Oncol. 26:329-34. ef fects on migraine. Cephalalgia. 26(1):33-42.

27. Korinth D, Pacyna-Gengelbach M, Deutchmann N, Hatten - 40. Qian Y, Venkatraj J, Barhoumi R Pal R, Datta A, Wild JR, berger S, Bockmuhl U, Dietel M, Schroeder H-G, Donhuijsen K, Tiffany-Castiglioni E. 2007. Comparative non-cholinergic neu - Petersen I. 2005. Chromosomal imbalances in wood dust-re - rotoxic effects of paraoxon and diisopropyl fluorophosphates lated adenocarcinomas of the inner nose and their associations (DFP) on human neuroblastoma and astrocytoma cell lines. with pathological parameters. J Pathol. 207:207-15. Toxicol Appl Pharmacol. 219(2-3):162-71.

28. Kwak S-E, Kim J-E, Kim D-S, Won M-H, Choi H-C, Kim Y-I, 41. Rehner SA and Buckley EP. 2003. Isolation and character - Song H-K, Choi S-Y, Kang T-C. 2005. Differential effects of ization of microsatellite loci from the entomopathogenic fungus vigabatrin and zonisamide on the neuropeptide Y system in Beauveria bassiana (Ascomycota: Hypocreales). Mol Ecol the hippocampus of seizure prone gerbil. Neuropeptides Notes. 3:409-11. 39(5):507-13. 42. Rejman J, Wagenaar A, Engberts J, Hoekstra D. 2004. 29. Lavania UC, Yamamoto M, Mukai Y. 2003. Extended Characterization and transfection properties of lipoplexes chromatin and DNA fibers from active plant nuclei for high- stabilized with novel exhangeable polyethylene glycol-lipid res olution FISH. J Histochem Cytochem. 51(10):1249-53. conju gates. Biochem Biophys Acta. 1660:41-52.

30. Lewis SM, Veyrier A, Ungureanu NH, Bonnal S, Vagner S, 43. Rola R, Zou Y, Huang T-T, Fishman K, Baure J, Rosi S, Holcik M. 2007. Subcellular relocalization of a trans -acting Mil liken H, Limoli CL, Fike JR. 2007. Lack of extracellular factor regulates XIAP IRES-dependent translation. MBC superoxide dismutase (EC-SOD) in the microenvironment 18(4):1302-11. impacts radiation-induced changes in neurogenesis. Free Radi cal Biol and Med. 42:1133-45. 31. Liu Z, Zhang B, Wei F, Li M. 2005. Isolation and charac - terization of microsatellite loxi for the red panda Ailurus ful - 44. Sagan SM, Rouleau Y, Leggiadro C, Supekova L, Schultz gens . Mol Ecol Notes 5:27-29. PG, Su AI, Pezacki JP. 2006. The influence of cholesterol, and lipid metabolism on host cell structure and hepatitis C virus 32. Lonskaya I, Potaman VN, Shlyakhtenko LS, Oussatcheva replication. Biochem Cell Biol. 84:67-79. EA, Lyubchenko YL, Soldatenkov VA. 2005. Regulation of poly(ADP-ribose) polymerase-1 by DNA structure-specific 45. Sakurai F, Inoue R, Nishino Y, Okuda A, Matsumoto O, bind ing. J Biol Chem. 280(17):17076-83. Taga T, Yanashita F, Takakura Y, Hashida M. 2000. Effect of DNA/liposome mixing ratio on the physicochemical characteris - 33. Marsden S, Nardelli M, Linder P, McCarthy JEG. 2006. tics, cellular uptake and intracellular trafficking of plasmid Unwinding single RNA molecules using helicases involved in DNA/cationic liposome complexes and subsequent gene ex - eukaryotic translation initiation. JMB. 361(2):327-35. pression. J Control Release. 66:255-69.

34. Menze MA, Hutchinson K, Laborde SM, Hand S. 2005. 46. Sambrook J, Fritsch EF, and Maniatis T. 1989. Molecular Mitochondrial permeability transition in the crustacian Artemia Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor franciscana : absence of a calcium-regulated pore in the face of Laboratory Press, Cold Spring Harbor, NY. profound calcium storage. Am J Physiol Regul Integr Comp Physiol. 289:68-76. 47. Sato T, Furukawa K. 2007. Sequential action of ETS-1 and SP1 for gene-activation of human ß-1,4-galactosyltrans - 35. Nan X, Tonary AM, Stolow A, Xie XS, Pezacki JP. 2006. ferase V involved in abnormal glycoslyation characteristic of Intracellular imaging of HCV RNA and cellular lipids by using cancer cells. J Biol Chem. 282:27702-12. si multaneous two-photon fluorescence and coherent anti- Stokes Raman scattering microscopies. ChemBioChem. 48. Savlik M, Polackova L, Szotakova B, Lamka J, Velik J, 7:1895-97. Skalova L. 2007. Activities of biotransformation enzymes in pheasant ( Phasianus colchicus ) and their modulation by in vivo 36. Noestheden M, Hu Q, Tonary AM, Tay L-L, Pezacki JP. administration of mebendazole and flubendazole. Res Vet Sci. 2007. Evaluation of chemical labeling strategies for monitoring 83:20-26. HCV RNA using vibrational microscopy. Org Biomol Chem. 5:2380-89. 49. Shimada S, Sunagawa M, Hanashiro K, Nakamura M, Ko sugi T. 2007. RNA interference targeting embryonic 37. Okuda K, Nakamura T, Sugita M, Shimizu T, Shikanai T. myosin heavy chain isoform inhibited mRNA expressions of 2006. A pentatricopeptide repeat protein is a site recognition phenotype markers in rabbit cultured vascular smooth muscle factor in chloroplast RNA editing. J Biol Chem. cells. Heart Vessels. 22:41-47. 281(49):37661-67. 50. Speranza P, Malesetti M. 2007. Nuclear and cytoplasmic 38. Ponnuraj EM, Hayward AR. 2002. Requirement for TNF- microsatellite markers for the species of the Dilatata group of Tnfrsf1 signalling for sclerosing cholangitis in mice chronically Paspalum (Poaceae). Plant Genetic Resources: Characterization infected by Cryptosporidium parvum . Clin Exp Immunol. and Utilization 5(1):14-26. 128:416-20.

www..vectorllabs..com 51. Swaroop A, Xu J, Agarwal N, Weissman SM. 1991. A simple and efficient cDNA library subtraction procedure: isola - tion of human retina-specific cDNA clones. Nucleic Acids Res. 19:1954.

52. Terhune SS, Schroer J, Shenk T. 2004. RNAs are packaged into human cytomegalovirus virions in proportion to their intra - cellular concentration. J Virol. 78(19):10390-98.

53. Tremblay JJ, Viger RS. 2003. Transcription factor GATA- 4 is activated by phosphorylation of serine 261 via the cAMP/Protein Kinase A signaling pathway in gonadal cells. J Biol Chem. 278:22128-35.

54. Trillo-Pazos G., Diamanturos A, Rislove L, Menza T, Chao W, Belem P, Sadiq S, Morgello S, Sharer L, Volsky DJ. 2003. Detection of HIV-1 DNA in microglia/macrophages, astrocytes and neurons isolated from brain tissue with HIV-1 encephalitis by laser capture microdissection. Brain Pathol. 13:144-54.

55. Tsang SH, Woodruff ML, Janisch KM, Cilluffo MC, Farber DB, Fain GL. 2007. Removal of phosphorylation sites of γ sub - unit of phosphodiesterase 6 alters rod light response. J Physiol. 579(2):303-12.

56. Wemmert S, Romeike BFM, Ketter R, Steudel W, Zang KD, Urbschat S. 2006. Intratumoral genetic heterogeneity in pilo - cytic astrocytomas revealed by CGH-analysis of microdissected tumor cells and FISH on tumor tissue section. Int J Oncol. 28:353-60.

57. Wettstein PJ, Borson ND, Park, JG, McNallan KT, Reed AM. 2005. Cysteine-tailed class I-binding peptides bind to CpG adjuvant and enhance primary CTL responses. J Immunol. 175: 3681-89.

58. Wiethoff CM, Koe JG, Koe GS, Middaugh CR. 2004. Compositional effects of cationic lipid/DNA delivery systems on transgene expression in cell culture. J Pharm Sci. 93(1):108-23.

59. Will C, Schewe C, Schluns K, Petersen I. 2006. HPV typing and CGH analysis for the differentiation of primary and metastatic squamous cell carcinomas of the aerodigestive tract. Cellular Oncol. 28(3):97-105.

60. Wu WWH, Sun Y-HB, Panté N. 2007. Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein. Virology J. 4:49-60.

61. Yasuda K, Ogawa Y, Yamane I, Nishikawa M, Takakura Y. 2005. Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9-dependent and –independent pathways. J Leukocyte Biol. 77:71-9.

62. Zaonarivelo JR, Sommer JA, Shore GE, McGuire SM, En gberg SE, Brienneman RA, Louis EE. 2007. Isolation and characterization of 20 microsatellite marker loci from the Indri ( Indri indri ) genome. Mol Ecol Notes 7(1):25-28. 32

ORDERING (800) 22 7-6666 (650) 69 7-3600 Fax: (650) 69 7-0339 (Order Department) ADDRESS: International orders are shipped by air. For the conven - 30 Ingold Road ience of our customers in the U.K. and Canada, Vector Burlingame, CA 94010 Laboratories maintains a complete inventory of all Vec tor products at our subsidiaries: TECHNICAL SERVICE: TEL: (650) 697-3600 Vector Laboratories Ltd., 3 Accent Park, Bakewell Road, Orton Southgate, Peterborough, PE2 6XS, FAX: (650) 697-0339 United Kingdom WEBSITE: http://www.vectorlabs.com tel (01733) 237999, fax (01733) 237119 email: [email protected] EMAIL: [email protected] Vector Laboratories (Canada), Inc. Outside USA: [email protected] 3390 South Service Road Orders may be placed by mail, telephone, fax or online. Burlington, Ontario, L7N 3J5, Canada The following must be included with each order: tel (905) 681-0900, fax (905) 681-0166 tel (888) 629-2121 (orders only) • Name of product and catalog number email: [email protected] • Quantity and size of items • Billing and shipping addresses Vector products are also available from over 40 • Purchase order number distributors worldwide. Please contact us or visit • Name and phone number of person placing our website for a complete listing of these repre - the order sentatives.

To place telephone orders call 650-697-3600 or toll For technical questions or more detailed information free 800-227-6666 from 8:30 am until 5:00 pm Pacific about our products, please contact our technical Time. For the convenience of our customers, Vector serv ice department. also accepts orders using VISA or Mastercard. Tele - Although provided in a highly purified form, our products are not intended phone orders over $1500 may require written/ faxed for clinical diagnosis or drug use, nor have they been packaged under sterile conditions. All products listed in this catalog are for research purposes only. confirmation. A confirmation should be boldly marked The listing of any product in this catalog does not imply the absence of a “Confirming Order. Do Not Duplicate.” Duplicate ship - patent covering its use, does not constitute license under any existing or pending patent, nor is it intended or implied as a recommendation for the ments due to incorrectly marked con firming orders use of such products in infringement of any patent. The responsibility for de termining the existence of such patents rests solely with the user. cannot be returned for credit. No returned product will be accepted or credited without prior authoriza - Trademarks of Vector: Animal-Free Blocker, AT-Yellow, DuoLuX, Elite, End - Tag, FastTag, GC-Red, HYBEX, NicKit p.s.o., Photoprobe, PolyBlock, ProtOn, tion from Vector Laboratories. Quant*Tag, Resolve-It, UltraSNAP, VectaMount, Vectashield, Vec tashield Hard !Set, Vectastain, Vector, Vector NovaRED, Vector Red, Vectrex. Others: Discounts for most products are available for large Texas Red-Molecular Probes; TSA-NEN Life Science Products; APS-5-Lumigen Inc. DuoLuX ™ is a special formula of APS-5 produced for Vector Labs by quantities or bulk orders. Orders for 10-29 of the Lu migen Inc. Southfield, MI. same product size will automatically receive a 10%

discount, orders for 30 or more will receive a 20% ® ®

® dis count. Please inquire for pricing of bulk quantities ® of any product.

Our payment terms are net 30 days. Prices are FCA Burlingame, California. Shipping charges will be prepaid and added to the invoice. Orders are usually shipped the same day they are received. Unless re quested otherwise, all products are shipped 2nd Day Air.

www..vectorllabs..com The text of this brochure has E been printed on recycled paper ©Vector Laboratories, Inc. 2007 Vector Laboratories, Inc. • 30 Ingold Road • Burlingame California 94010 USA PH: (650) 697-3600 FAX (650) 697-0339 For Orders: (800) 227-6666 EMAIL: [email protected] Vector Laboratories, Ltd. • 3 Accent Park, Bakewell Road • Orton Southgate, Peterborough PE2 6XS England UK PH: (01733) 237999 FAX (01733) 237119 EMAIL: [email protected] Vector Laboratories, (Canada) Inc. • 3390 South Service Road • Burlington, Ontario L7N 3J5 Canada PH: (905) 681-0900 FAX (905) 681-0166 For Orders: (888) 629-2121 EMAIL: [email protected]